Compositions and methods for administration of vaccines against dengue virus

ABSTRACT

Embodiments of the present invention report compositions and methods for vaccinating a subject against dengue viruses. In some embodiments, vaccine compositions may be administered by intradermal introduction. In certain embodiments, intradermal introduction in a subject of a vaccine against dengue virus may include one or more intradermal boosts after initial vaccination. Other embodiments include intradermal injection of a vaccine composition against dengue virus wherein the composition provides protection against two or more of DEN-1, DEN-2, DEN-3 and DEN-4.

PRIORITY

This application claims the benefit under 35 USC §119(e) of provisionalU.S. patent application Ser. No. 61/183,020 filed on Jun. 1, 2009, whichis incorporated herein by reference in its entirety.

FEDERALLY FUNDED RESEARCH

Embodiments disclosed herein were supported in part by a grant from theNational Institutes of Allergy and Infectious Diseases, a division ofthe National Institute of Health, Grant Number 5U01A1070443. The U.S.government may have certain rights to practice the subject invention.

FIELD

Embodiments of the present invention report compositions and methods foradministering a vaccine to a subject against dengue viruses. In someembodiments, vaccine compositions may be administered by intradermalinjection. In certain embodiments, intradermal injection in a subject ofa vaccine against dengue virus may include one or more intradermalboosts after initial vaccination. Other embodiments include intradermalinjection of a vaccine composition against dengue virus wherein thecomposition provides protection against more than one serotype of denguevirus, such as DEN-1, DEN-2, DEN-3 and DEN-4.

BACKGROUND

Vaccines for protection against viral infections have been effectivelyused to reduce the incidence of human disease. One of the mostsuccessful technologies for viral vaccines is to immunize animals orhumans with a weakened or attenuated strain of the virus (a “live,attenuated virus”). Due to limited replication after immunization, theattenuated strain does not cause disease. However, the limited viralreplication is sufficient to express the full repertoire of viralantigens and can generate potent and long-lasting immune responses tothe virus. Thus, upon subsequent exposure to a pathogenic strain of thevirus, the immunized individual is protected from disease. These live,attenuated viral vaccines are among the most successful vaccines used inpublic health.

SUMMARY

Embodiments of the present invention generally relate to methods andcompositions for inducing protection in a subject against dengue virusby, for example, administering a vaccine to a subject against dengueviruses. Some embodiments can include introducing a vaccine compositionto a subject via intradermal (ID) injection. In accordance with theseembodiments, the vaccine composition can be introduced to a subjectintradermally to, for example, protect against one or more than onedengue serotype (e.g. cross protection). In certain embodiments, avaccine composition can include, but is not limited to, a single dose ofone serotype of dengue virus (e.g. DENVax 4) administered to a subject.In other embodiments, a vaccine composition may include, but is notlimited to; an initial dose of one serotype of dengue virus (e.g. DENVax4 or other serotype) and then one or more boosts of the same, acombination or a different serotype can be administered to a subject.

Other aspects herein can concern inducing a cellular immune response ina subject by, for example, introducing a vaccine composition to asubject via intradermal introduction wherein the vaccine compositionincludes, but is not limited to, a dengue virus vaccine. In accordancewith these embodiments, compositions disclosed can be administeredintradermally to a subject for modulating neutralizing antibodyproduction in the subject against dengue virus serotypes. Some aspectsconcern predetermined composition ratios (e.g. 1:1, 1:2, 1:4, any ratioof two or more serotypes is contemplated) of the various serotypes ofdengue virus or fragments thereof or attenuated compositions thereof ina single vaccine composition in order to increase cross protection in asubject against some or all dengue virus serotypes when the subject isadministered the single vaccine composition intradermally.

In certain embodiments, some advantages of using intradermalintroduction of a vaccine against dengue virus can include, but are notlimited to, multiple protection against some or all dengue virusserotypes in a subject, reduced cost by using small doses compared tosubcutaneous injection, modulation of antibodies produced against someor all dengue virus serotypes in a subject and reduced pain at a site ofadministration in a subject administered a composition of vaccineagainst dengue virus.

In some embodiments, a single dose vaccine against dengue virus caninclude one or more dengue virus serotype(s). In accordance with theseembodiments, a subject may be treated with at least one additionalintradermal injection(s) administered at a separate site from the firstinjection, for example, next to or in a separate anatomical site on thesubject. In addition, at least one additional intradermal injection(s)may be performed less than 30 days after the first administration to thesubject. Vaccine compositions of these and other embodiments disclosedherein may include two or more dengue, virus serotypes at apredetermined ratio.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain embodiments. Some embodimentsmay be better understood by reference to one or more of these drawingsalone or in combination with the detailed description of specificembodiments presented.

FIG. 1 represents an example of an intradermal injection devicecurrently available.

FIG. 2 represents examples of injection sites in a subject havingintradermal introduction of a vaccine against dengue virus.

FIG. 3 represents a bar graph comparison of subcutaneous versusintradermal injection of a vaccine against dengue virus and neutralizingantibody titer produced against different dengue virus serotypes after aprimary administration.

FIG. 4 represents a bar graph comparison of subcutaneous versusintradermal injection of a vaccine against dengue virus and neutralizingantibody titer produced against different dengue virus serotypes after asecond, boosting administration.

FIG. 5 represents a histogram plot of subcutaneous and intradermalimmunizations with a vaccine against a dengue virus serotype in mice.

FIGS. 6A and 6B represent graphic depictions of a challenge experimentusing two different dengue virus serotypes on a dengue serotype-immunemouse population following vaccination of the mice with another denguevirus serotype.

DEFINITIONS

As used herein, “a” or “an” may mean one or more than one of an item.

As used herein, vessel can include, but is not limited to, test tube,mini- or micro-fuge tube, channel, vial, microtiter plate or container.

As used herein the specification, “subject” or “subjects” may includebut are not limited mammals such as humans or mammals, domesticated orwild, for example dogs, cats, other household pets (e.g., hamster,guinea pig, mouse, rat), ferrets, rabbits, pigs, horses, cattle, prairiedogs, or zoo animals.

As used herein, “about” can mean plus or minus ten percent.

As used herein, “attenuated virus” can mean a virus that demonstratesreduced or no clinical signs of disease when administered to a subjectsuch as a mammal (e.g., human or an animal).

DESCRIPTION

In the following sections, various exemplary compositions and methodsare described in order to detail various embodiments. It will be obviousto one skilled in the art that practicing the various embodiments doesnot require the employment of all or even some of the specific detailsoutlined herein, but rather that concentrations, times and otherspecific details may be modified through routine experimentation. Insome cases, well-known methods or components have not been included inthe description.

Certain aspects of the present invention include, but are not limitedto, administration of vaccine compositions against dengue virus.

Embodiments of the present invention generally relate to methods andcompositions for inducing protection in a subject against dengue virusserotypes. Other embodiments can include introducing a vaccinecomposition to a subject via intradermal (ID) injection wherein thevaccine composition introduced intradermally induces cross protectionagainst some or all dengue serotypes. In certain embodiments, thevaccine composition comprises a single dose of a vaccine against denguevirus serotype 4 (DENVax 4) administered to a subject. In otherembodiments, the vaccine composition comprises an initial dose of DENVax4 then, one or more boosts of the vaccine administered to a subject.

Other aspects of the present invention include modulating an immuneresponse to a vaccine against dengue virus administered intradermallycompared to subcutaneously to a subject. Vaccines against dengue virusmay include a composition comprising ratios of serotypes of denguevirus, live attenuated dengue virus, or fragments thereof such asproteins or nucleic acids derived or obtained from dengue virusserotypes. Ratios of various serotypes may be equal or certain serotypesmay be represented more than others depending on need or exposure orpotential exposure to the virus. In accordance with these embodiments, aratio may be a 1:2, 1:3, 1:4, 1:10, 1:20; 1:1:1, 1:2:2, 1:2:1, 1:1:1:1,1:2:1:2; 1:3:1:3, 2:3:3:3, 5:4:5:5, 1:2:2 or any ratio for any ofserotypes 1, 2, 3 and/or 4, depending on for example, number ofserotypes represented in the formulation, predetermined response andeffect desired. It is contemplated that any dengue virus serotypeformulation may be used to generate a vaccine (e.g. attenuated virusetc.) of use in intradermal administration to a subject in need thereof.It is contemplated that some formulations may be more effective thanothers when introduced intradermally than other formulations.

In other embodiments, compositions of dengue virus vaccine formulationsmay be introduced intradermally to a subject prior to, during or afterexposure to dengue virus by the subject. In accordance with theseembodiments, a subject may receive a single intradermal injection ormore than one injection comprising a dengue virus formulation,optionally, followed by one or more additional injections. Intradermalapplications of formulations described herein may be combined with anyother anti-viral treatment or administration mode of vaccine (e.g.subcutaneous injection) to a subject. In some embodiments, it iscontemplated that intradermal introduction of a formulation contemplatedherein may be administered to any appropriate region of a subject's body(e.g. arm, hip, etc). In addition, intradermal administration of vaccineformulations herein as primary or boost administrations may occur in thesame day, consecutive days, weekly, monthly, bi-monthly or otherappropriate treatment regimen.

Methods Nucleic Acid Amplification

Nucleic acids may be used in any formulation or used to generate anyformulation contemplated herein. Nucleic acid sequences used as atemplate for amplification can be isolated viruses (e.g. dengueviruses), according to standard methodologies. A nucleic acid sequencemay be genomic DNA or fractionated or whole cell RNA. Where RNA is used,it may be desired to convert the RNA to a complementary cDNA. In someembodiments, the RNA is whole cell RNA and is used directly as thetemplate for amplification. Any method known in the art for amplifyingnucleic acid molecules is contemplated (e.g., PCR, LCR, Replicase, etc).

Expressed Proteins or Peptides

Genes can be expressed in any number of different recombinant DNAexpression systems to generate large amounts of the polypeptide product,which can then be purified and used in methods and compositions reportedherein. Any method known in the art for generating and using constructsis contemplated. In certain embodiments, genes or gene fragmentsencoding one or more polypeptide may be inserted into an expressionvector by standard cloning or subcloning techniques known in the art.

Proteins, peptides and/or antibodies or fragments thereof may bedetected or analyzed by any means known in the art. In certainembodiments, methods for separating and analyzing molecules may be usedsuch as gel electrophoresis or column chromatography methods.

Electrophoresis

Electrophoresis may be used to separate molecules (e.g., large moleculessuch as proteins or nucleic acids) based on their size and electricalcharge. There are many variations of electrophoresis known in the art. Asolution through which the molecules move may be free, usually incapillary tubes, or it may be embedded in a matrix or other materialknown in the art. Common matrices can include, but are not limited to,polyacrylamide gels, agarose gels, mass spec, blotting and filter paper.

Some embodiments, using a gene or gene fragment encoding a polypeptidemay be inserted into an expression vector by standard subcloningtechniques. An expression vector may be used which produces therecombinant polypeptide as a fusion protein, allowing rapid affinitypurification of a peptide or protein. Examples of such fusion proteinexpression systems are the glutathione S-transferase system (Pharmacia,Piscataway, N.J.), the maltose binding protein system (NEB, Beverley,Mass.), the FLAG system (IBI, New Haven, Conn.), and the 6×His system(Qiagen, Chatsworth, Calif.).

Pharmaceutical Formulations

Any pharmaceutical formulation known in the art for a vaccine iscontemplated herein. In certain embodiments, a formulation can containone or more DEN serotype in various ratios, depending on predeterminedexposure, to or existence of dengue virus subtypes. It is contemplatedthat formulations can contain other agents of use in vaccination of asubject including, but not limited to other active, or inactiveingredients or compositions known to one skilled in the art.

Kits

Other embodiments concern kits of use with the methods (e.g. methods ofapplication or administration of a vaccine) and compositions describedherein. Some embodiments concern kits having vaccine compositions of useto prevent or treat subjects having, exposed or suspected of beingexposed to one or more dengue viruses. In certain embodiments, a kit maycontain one or more than one formulation of dengue virus serotype(s)(e.g. attenuated vaccines) at predetermined ratios. Kits can beportable, for example, able to be transported and used in remote areassuch as military installations or remote villages. Other kits may be ofuse in a health facility to treat a subject having been exposed to oneor more dengue viruses or suspected of being at risk of exposure todengue virus.

Kits can also include a suitable container, for example, vials, tubes,mini- or microfuge tubes, test tube, flask, bottle, syringe or othercontainer. Where an additional component or agent is provided, the kitcan contain one or more additional containers into which this agent orcomponent may be placed. Kits herein will also typically include a meansfor containing the agent, composition and any other reagent containersin close confinement for commercial sale. Such containers may includeinjection or blow-molded plastic containers into which the desired vialsare retained. Optionally, one or more additional agents such asimmunogenic agents or other anti-viral agents, anti-fungal oranti-bacterial agents may be needed for compositions described, forexample, for compositions of use as a vaccine against one or moreadditional microorganisms.

The following examples are included to demonstrate certain embodimentspresented herein. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples that follow representtechniques discovered to function well in the practices disclosedherein. However, those of skill in the art should, in light of thepresent disclosure, appreciate that many changes can be made in thecertain embodiments which are disclosed and still obtain a like orsimilar result without departing from the spirit and scope herein.

EXAMPLES Example 1

Previous studies revealed that natural infection with each DEN (denguevirus) serotype leads to long-lived protection against dengue levercaused by the homologous serotype. In certain embodiments,administration of an effective dengue vaccine closely mimics naturalinfection and can serve as a mode for administering vaccines againstdengue virus. Embodiments reported herein can concern a naturalinfection route of dengue virus (DEN) infection, similar to intradermaldelivery by the transporting host, a mosquito bite. In certainembodiments, intradermal injection to deposit the vaccine viruses intothe same tissue can be used. Skin is a highly accessible organ andrepresents an effective immune barrier, mainly attributed to thepresence of Langerhans cells (LCs) residing in the epidermis. Skinimmunization elicits a broad range of immune responses, includinghumoral, cellular, and mucosal and has the potential to bypass theeffect of pre-existing immunity on the immunogenicity of administeredvaccines.

Some embodiments for intradermal (ID) administration of the tetravalentdengue vaccines in a subject in need of such a treatment are reported.One exemplary method of intradermal administration was performed on fourCynomolgous macaques administered a 5:5:5:5 DENVax (dengue virusvaccine) by intradermal administration. To achieve an equivalent dose ofvirus, 0.15 ml of vaccine was deposited. ID in three closely spacedsites using a needle-free jet injector (see FIGS. 1 and 2, below), FIG.1 represents an intradermal inject (e.g., PharmaJet®) device used forintradermal inoculations.

FIG. 2 illustrates inoculation sites on Cynomolgus macaques postvaccination with PharmaJet device. Animals were boosted 60 days laterwith the same formulation by the same route. Serum samples werecollected at predetermined intervals, days 15, 30, 58, 74, and 91 andwere tested for the presence of neutralizing antibodies directed againstthe four dengue serotypes. PRNT (plaque reduction neutralization test,known in the art for quantifying levels of anti-DEN neutralizingantibodies) were performed on the sera samples.

It was demonstrated that the neutralizing antibody titers aresignificantly higher after ID administration as compared to SCadministration after a primary administration (see FIG. 3) or after asecondary administration (See FIG. 4). Since the sites were closelyspaced in the same area, and each inoculum consists of all four viruses,this mode of vaccine delivery closely resembles a single administrationof DENVax. FIG. 3 illustrates 50% PRNT Geometric Mean Titers at Day 58(58 days after the primary administration). FIG. 4 illustrates 50% PRNTGeometric Mean Titers at Day 74 (14 days after the secondaryadministration on Day 60). As can be seen in the figures, theneutralizing antibody titers to all four dengue viruses were higherafter intradermal versus subcutaneous administration. In addition, thenumber of animals that demonstrated neutralizing antibody responses(“seroconversion” defined as PRNT>10) was greater after the first doseof vaccine (see Table 1, the percentage of animals that seroconverted toeach of the four dengue serotypes is shown after primary and secondaryimmunization).

TABLE 1 Seroconversion of non-human primates after dengue immunization %Seroconversion DENVax DEN-1 DEN-2 DEN-3 DEN-4 Formyiation Prime BoostPrime Boost Prime Boost Prime Boost 5:5:5:5 SC 87.5% 100.0% 100.0%100.0% 75.0% 100.0% 50.0% 100.0% 5:5:5:5 ID 100.0% 100.0% 100.0% 100.0%100.0% 100.0% 100.0% 100.0%

The immunized animals were tested for protection against challenge withwild type dengue viruses. In cynomolgus macaques, wild type dengue virusinfection leads to virus replication and viremia, but no clinical signs.At day 91, two monkeys were challenged with DENV-1 (dengue virusserotype 1) and two monkeys challenged with DEN-2 (dengue virus serotype2). Serum samples were collected daily for 11 days after challenge.Levels of dengue virus RNA were measured in the samples by quantitativereal-time polymerase chain reaction technology (q-rtPCR) and titers ofviable virus were measured by virus isolation and plaque formation onVero cells. The results are shown in Tables 2 and 3. Neutralizingantibodies against DEN-1 at Day 91, just prior to challenge(“Pre-Challenge”) and Day 105, 14 days after challenge (“Post”). Viremiais given as the number of days that live DEN-1 virus could be isolatedfrom blood samples (“Duration”) and the log 10 of the peak titerisolated from each animal. Viral RNA is given as the number of daysviral RNA could be detected in the serum samples (“Duration”) and peakviral RNA levels in each monkey, expressed as the log 10 of the numberof viral RNA genomes detected.

TABLE 2 Responses after challenge with DEN-1 DEN-1 PRNT Viremia ViralRNA Monkey Formulation Pre-Challenge Post Duration Peak Duration PeakCY0174 5:5:5:5 SC 240 240 0 0 0 0 CY0181 5:5:5:5 SC 640 61440 0 0 5 5.6CY0192 5:5:5:5 ID 1920 1280 0 0 0 0 CY0194 5:5:5:5 ID 7680 1920 0 0 0 0CY0061 Controls 1 2560 6 2.0 9 5.7 CY0193 Controls 25 2580 3 2.7 7 6.4CY0058 Controls 1 640 5 2.9 7 5.5 CY0073 Controls 1 1280 5 3.6 10 6.2

TABLE 3 Responses after challenge with DEN-2 DEN-2 PRNT Viremia ViralRNA Monkey Formulation Pre-Challenge Post Duration Peak Duration PeakCY0172 5:5:5:5 SC 3413 3413 0 0 1 3.9 CY0177 5:5:5:5 SC 853 533 0 0 0 0CY0198 5:5:5:5 ID 240 320 0 0 0 0 CY0201 5:5:5:5 ID 1920 1600 0 0 0 0CY0088 Controls 6 10240 6 2.3 8 5.1 CY0199 Controls 1 3840 5 1.8 9 4.7CY0065 Controls 1 10240 5 2.9 8 5.8 CY0104 Controls 1 10240 4 2.4 8 5.7

After challenge, the SC and ID immunized animals were completelyprotected from DEN-1 or DEN-2 induced viremia (compared to the controlanimals that demonstrated significant viremia of long duration). In allof the ID immunized animals, but not all of the SC immunized animals,there was also an absence of viral RNA replication and a lack of anincrease in antibody titer after challenge (compare the ID animals to SCinjected CY0181, CY0.172 or the control animals). These data suggestthat protection is “sterilizing” and prevents any virus replicationafter challenge.

Example 2

in another example, an optimized DENVax formulation delivered indifferent locations and with different timings will be tested innon-human primates, Groups of eight Cynomolgus macaques will beimmunized with a DENVax formulation containing 1×10⁵ plaque formingunits (pfu), 1×10⁴ pfu, 1×10⁵ pfu and 1×10⁵ pfu of DENVAx-1, DENVax-2,DENVax-3 and DENVax-4, respectively (abbreviated 5:4:5:5). Two doseswill be administered in 0.1 ml ID. Groups will be immunized with eitherone dose in each arm at Day 0, one dose in one arm at Day 0 and one dosein the other arm at Day 7, or one dose in one arm at Day 0 and one dosein the other arm at Day 60, These groups will be compared to a groupthat receives the same dose (5:4:5:5) in three sites in the same are onDay 0 and three sites in the other arm on Day 60 as well as a group thatreceives the same dose in a single 0.5 ml SC immunization in one arm atDay 0 and in the other arm at Day 60. A control group will be immunizedwith vaccine excipients only (no vaccine viruses). Followingimmunization, blood samples will be collected on days 0, 7 (for peakviremia), 15, 30, 60, and 90 to test the neutralizing antibodies againstthe four dengue virus serotypes by PRNT50. PBMCs collected on days 30,60, 90 will be also monitored for IFN-γ secretion by an ELISPOT assay.On day 90, two animals from each group will be challenged with wild typeof DEN-1, DEN-2, DEN-3, or DEN-4 viruses. Challenged animals will bemonitored for clinical signs and, temperature (twice daily), changes infood consumption (once daily) and body weight (weekly). In addition, allanimals will be bled daily for 11 days post-challenge to monitor viremiaand hematological parameters. Again, the speed and duration of PRNTresponses to all four DEN viruses and protection after day 90 challengewill be assessed. It is believed that intradermal administration inmultiple sites and in distinct anatomical locations may be moreeffective than subcutaneous administration as a single bolus. Multiplesites can provide exposure of the vaccine to more antigen presentingcells. Distinct anatomical locations can permit vaccine access tomultiple lymph nodes. In addition, booster immunizations of denguevaccines have only been administered after the development of antibodyresponses in mice, primates and human clinical trials, thirty days orlonger. At this time, neutralizing antibodies inhibit the response tothe live viral vaccines. It was previously shown that boosting primatesone month after primary immunization was less effective than dosing fourmonths after primary immunization. It was speculated that high levels ofhomologous and heterologous antibodies that circulate after the initialimmunization can inhibit viral replication in a second dose. Whileprolonged (two months or longer) immunization may circumvent thisinhibition, it has not been tested whether accelerated immunizationregimen with shorter immunization intervals, before the development ofpotent neutralizing antibody responses may be advantageous. Such ashortened regimen may be an advantage in endemic countries or fortravelers, where exposure to dengue viruses in between the immunizationsmay put them at risk of disease.

Example 3

In another example, a human clinical trial has been initiated, studyingthe safety and immunogenicity of two DENVax formulations, administeredin 0.1 ml either by ID or SC injection. Groups of 12 individuals will beimmunized with for example, a low dose DENVax formulation (8×10³ pfu,5×10³ pfu, 1×10⁴ pfu and 2×1 pfu of DENVax-1, -2, -3 and -4,respectively) or a high dose (2×10⁴ pfu, 5×10⁴ pfu, 1×10⁵ pfu and 3×10⁵pfu of DENVax-1, -2, -3 and -4, respectively) of DENVax ID or SC on Days0 and 90. Two control groups will be injected SC or ID withphosphate-buffered saline. Patients will be monitored for any adverseevents, and for any significant changes in hematological or bloodchemistry parameters. Serum samples will be collected to measure vaccinevirus replication and neutralizing antibody responses at periodicintervals.

Example 4

immunogenicity and efficacy of DENVax administered intradermally inAG129 mice. In another example, two studies were performed to comparethe effect of route of administration on immunogenicity and efficacy ofDENVax in AG129 mice. In one example, the immunogenicity of monovalentDENV^(a)x-4 (e.g. vaccine against one Dengue virus serotype) wascompared in AG129 mice by measuring the neutralizing antibody responsesfollowing SC injection under the skin on the back or ID injection intothe foot pad using a needle and syringe. Groups of 8 AG129 mice wereinjected ID or SC with 10⁵ PFU/dose of chimeric DENVax-4 vaccine in 50μl and 100 μl final volume, respectively. Six weeks after priming,animals from each treatment group were boosted via the corresponding IDor SC route with 10⁵ PFU of DENVax-4 or TEA. Mice were bled on Day 31and 58 and collected sera were pooled to measure neutralizing antibodyresponses.

Immunization of DENVax-4 via the ID route elicited a 5-fold higherneutralizing antibody response to DEN-4 after the boost compared to theresponse induced via the SC route (see for example, FIG. 4). Theanti-DEN-4 response elicited by either route of immunization had amarked cross-neutralizing activity against DEN-3 but not against DEN-1or DEN-2 serotypes. FIG. 4 represents neutralizing antibody responsesfollowing primary and secondary immunization of AG129 mice with chimericDENVax-4. Mice were bled on Day 31 and 58 and collected sera were pooledto measure neutralizing antibody responses using the plaque reductionassay (PRNT50).

Two weeks after the boost animals from each group were split in to twogroups and challenged with 10⁶PFU of DEN-1 (Mochizuki virus strain) orDEN-2 (New Guinea C strain) viruses. Challenged animals were monitoredfor clinical signs of disease and survival rates were recorded over aperiod of 5 weeks. Mice immunized via the ID route showed no signs ofdisease after DEN-1 challenge (FIG. 5A). In the SC immunized group onlyone mouse succumbed to infection while the rest of animals had no anyapparent signs of infection (FIG. 5B). In contrast, all control animalssuccumbed to infection by day 13 after DEN-1 challenge (FIG. 5A).Following DEN-2 challenge, all animals immunized with only DENVax-4 viathe SC route succumbed to infection by day 2.5 with mean survival time(MST) of 19.5 days as compared to the control (TFA) mice that allsuccumbed by day 17 (MST=12.5 days) post-challenge (FIG. 5B). Incontrast, fifty percent of ID DENVax-4 immunized mice survived theinfection until the cud of the 5 week monitoring period (FIG. 5B). FIGS.5A and 5 B represent survivals of DENVax-4 immune AG129 mice followingchallenge with DEN-1 (a) or DEN-2 (b) viruses. Challenged animals weremonitored for clinical signs of disease and survival rates were recordedover a period of 5 weeks.

In a second study, immunogenicity of tetravalent DENVax vaccineadministered SC or ID in mice (e.g. AG129) was tested. Groups of AG129mice, six per group were injected SC or ID with the DENVax in 100 μl or50 μl (final volume), respectively. Mice were immunized with DENVax at a5:4:5:5 (10⁵ PFU of DENVax-1, -3 and -4 and 10⁴ PFU of DENVax-2) doselevel of composite chimeric vaccines. All immunized animals received abooster injection of 5:4:5:5 DENVax 42 days' post-primary inoculation.Blood samples were collected on days 42 and 56 to measure neutralizingantibody responses to each DEN virus serotype.

As represented in Table 4, both primary and secondary neutralizingantibody responses to all four DEN serotypes were induced. Following theboost, the neutralizing anti-DEN-1, DEN-3 and DEN-4 antibody titers wereincreased by 2, 5 and 2 fold, respectively in the group of mice injectedID as compared to the SC immunized animals. Neutralizing responses toDEN-2 virus were comparable in both groups. Immunization via the SCroute resulted in a profile of dominant neutralizing antibody responsesagainst DEN-1>DEN-2>DEN-3>DEN-4, with neutralizing titers 5120, 1280,640 and 80, respectively. The hierarchy of neutralizing antibodyresponses after ID administration had shifted as follows;DEN-1>DEN-3>DEN-2>DEN-4 with neutralizing antibody titers 10240, 3840,1280 and 160, respectively.

TABLE 4 Comparison of the immunogenicity of tetravalent DENVax bearingthe ratio 5:4:5:5 PFU of each composite chimeric virus (10⁵ PFU ofDENVax-1, -3 and -4 and 10⁴ PFU of DENVax-2) after SC or ID immunizationof mice. Neutralizing Antibody Titers (GMT) DENVax DEN-1 DEN-2 DEN-3DEN-4 Formulation Prime Boost Prime Boost Prime Boost Prime Boost5:4:5:5/SC 1920 5120 3200 1280 1280 640 80 80 5:4:5:5/ID 2560 10240 12801280 1600 3840 120 160 Blood samples were collected on days 42 and 56 tomeasure neutralizing antibody responses to each DEN virus serotype.

Materials and Methods

Mice: AG129 mice have an “intact” immune system; deficient for theinterferon (IFN)-α/β and -γ receptors. Dengue infection has beendescribed. Other studies: pathogenesis, cell tropism, and ADE have beenexamined. This model permits challenge with DEN-1 and DEN-2.

Non-human primates: Cynomolgus, rhesus macaques carry virus (viremia),but no disease manifests.

All of the COMPOSITIONS and METHODS disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods have been described interms of preferred embodiments, it is apparent to those of skill in theart that variations maybe applied to the COMPOSITIONS and METHODS and inthe steps or in the sequence of steps of the methods described hereinwithout departing from the concept, spirit and scope herein. Morespecifically, certain agents that are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept as defined bythe appended claims.

1. A method for inducing protection in a subject against multiple denguevirus serotypes, comprising, administering a single dose vaccine againstdengue virus to a subject by intradermal introduction, inducingprotection in the subject against multiple dengue virus serotypes. 2.The method of claim 1, further comprising administering at least oneadditional intradermal injection(s) of the vaccine against dengue virus.3. The method of claim 1, wherein the single dose vaccine against denguevirus comprises one or more dengue virus serotype(s).
 4. The method ofclaim 1, wherein at least one additional intradermal injection(s) isperformed at a separate site on the subject.
 5. The method of claim 1,wherein at least one additional intradermal injection(s) is performedless than 30 days after the first administration to the subject.
 6. Themethod of claim 1, wherein the vaccine comprises at least two denguevirus serotypes at a predetermined ratio.
 7. The method of claim 1,wherein the vaccine comprises all four dengue virus serotypes.
 8. Themethod of claim 1, further comprising administering at least oneimmunogenic agent to the subject.
 9. The method of claim 8, wherein theimmunogenic agent comprises at least one Toll receptor (TLR) ligand(s).10. A composition for protecting a subject against a dengue virusserotype comprising, an intradermally (ID) administered single dosecomposition of predetermined ratio of at least 2 dengue virus serotypes.11. The composition of claim 10, wherein the compositions is 10% or morereduced in concentration compared to the composition administeredsubcutaneously in a range of 10² to 10⁵ pfu of one or more liveattenuated dengue vaccines.
 12. The composition of claim 10, wherein thecomposition comprises all 4 dengue virus serotypes.
 13. The compositionof claim 12, wherein the 4 serotypes are in a predetermined ratio. 14.The composition of claim 10, further comprising at least one immunogenicagent.
 15. The composition of claim 10, further comprising at least oneToll Receptor (TLR) ligand(s) or other adjuvant.
 16. The composition ofclaim 15, wherein the at least one Toll Receptor ligand(s) comprisesCpG-ODN, Poly I:C, imiquimod, inulin-derived adjuvants, MPL a Tollreceptor 4 ligand, poly I:U a Toll receptor 3 ligand, heat labileenterotoxin, non toxic mutants of heat labile enterotoxin and acombination thereof.
 17. A system for administering a dengue vaccinecomposition to a subject comprising, a vaccine composition againstdengue virus and an apparatus capable of introducing intradermally thevaccine composition by a non-injectible method to a subject,administering the dengue vaccine composition to the subject.
 18. Thesystem of claim 17, wherein the non-injectible method is without needlesand without syringes.
 19. A vaccine kit comprising; at least one vaccinecomposition against dengue virus comprising one or more dengue virusserotypes; and at least one apparatus capable of intradermaladministration of the composition to a subject.
 20. The kit of claim 19,further comprising at least one immunogenic agent.
 21. The kit of claim19, further comprising at least one Toll Receptor ligand(s).